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Competitive amplification of differentially melting amplicons (CADMA) improves KRAS hotspot mutation testing in colorectal cancer.

ABSTRACT: Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC) a selected group of patients with wild-type KRAS respond to antibodies against the epidermal growth factor receptor (EGFR). Testing for KRAS mutations is now required prior to anti-EGFR treatment, however, less sensitive methods based on conventional PCR regularly fail to detect KRAS mutations in clinical samples.We have developed sensitive and specific assays for detection of the seven most common KRAS mutations based on a novel methodology named Competitive Amplification of Differentially Melting Amplicons (CADMA). The clinical applicability of these assays was assessed by analyzing 100 colorectal cancer samples, for which KRAS mutation status has been evaluated by the commercially available TheraScreen® KRAS mutation kit.The CADMA assays were sensitive to at least 0.5% mutant alleles in a wild-type background when using 50 nanograms of DNA in the reactions. Consensus between CADMA and the TheraScreen kit was observed in 96% of the colorectal cancer samples. In cases where disagreement was observed the CADMA result could be confirmed by a previously published assay based on TaqMan probes and by fast COLD-PCR followed by Sanger sequencing.The high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.

SUBMITTER: Kristensen LS

PROVIDER: S-EPMC3517778 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Competitive amplification of differentially melting amplicons (CADMA) improves KRAS hotspot mutation testing in colorectal cancer.

Kristensen Lasse Sommer LS Kjeldsen Tina Ellegaard TE Hager Henrik H Hansen Lise Lotte LL

BMC cancer 20121123

<h4>Background</h4>Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC) a selected group of pa ...[more]

PMID: 23173730

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Project description:BackgroundKRAS mutational analysis is the standard of care prior to initiation of treatments targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer. Sensitive methods are required to reliably detect KRAS mutations in tumor samples due to admixture with non-mutated cells. Many laboratories have implemented sensitive tests for KRAS mutations, but the methods often require expensive instrumentation and reagents, parallel reactions, multiple steps, or opening PCR tubes.MethodsWe developed a highly sensitive, single-reaction, closed-tube strategy to detect all clinically significant mutations in KRAS codons 12 and 13 using the Roche LightCycler® instrument. The assay detects mutations via PCR-melting curve analysis with a Cy5.5-labeled sensor probe that straddles codons 12 and 13. Incorporating a fast COLD-PCR cycling program with a critical denaturation temperature (Tc) of 81°C increased the sensitivity of the assay >10-fold for the majority of KRAS mutations.ResultsWe compared the COLD-PCR enhanced melting curve method to melting curve analysis without COLD-PCR and to traditional Sanger sequencing. In a cohort of 61 formalin-fixed paraffin-embedded colorectal cancer specimens, 29/61 were classified as mutant and 28/61 as wild type across all methods. Importantly, 4/61 (6%) were re-classified from wild type to mutant by the more sensitive COLD-PCR melting curve method. These 4 samples were confirmed to harbor clinically-significant KRAS mutations by COLD-PCR DNA sequencing. Five independent mixing studies using mutation-discordant pairs of cell lines and patient specimens demonstrated that the COLD-PCR enhanced melting curve assay could consistently detect down to 1% mutant DNA in a wild type background.ConclusionsWe have developed and validated an inexpensive, rapid, and highly sensitive clinical assay for KRAS mutations that is the first report of COLD-PCR combined with probe-based melting curve analysis. This assay significantly improved diagnostic accuracy compared to traditional PCR and direct sequencing.

Project description:KRAS mutation testing helps oncologists decide whether patients diagnosed with metastatic colorectal cancer (CRC) should be treated with epidermal growth factor receptor (EGFR) inhibitors. These drugs block the EGFR signaling pathway in tumor cells, and thus can slow tumor progression. Mutated KRAS is found in 30?45% of all CRC tumors, with mutations occurring at codons 12 and 13 being the most common. Patients with a mutated KRAS found in tumor tissue show limited clinical response to EGFR inhibitor therapy. Several factors can influence the results of KRAS mutation testing in tumor specimens. Variation can result from tumor heterogeneity, sample handling, DNA preparation, and differences in assay design and methodology. The purpose of this study was to evaluate comparability of KRAS test results among five labs currently used to determine KRAS mutation status of colorectal cancer specimens in a large multi-center study.Three commercial labs (Genzyme, Clarient, Quest Diagnostics), one clinical lab (Henry Ford Health System), and one research lab (Oregon Health Sciences University) were contracted to analyze KRAS mutational status; all are Clinical Laboratory Improvement Amendments (CLIA) certified. Twenty formalin-fixed paraffin-embedded (FFPE) human CRC samples that had been previously tested for KRAS mutations were selected based on mutation status (7 wild type samples, 7 with codon 12 mutations, and 6 with codon 13 mutations) from two clinical settings (Kaiser Permanente Colorado and Northwest). To ensure sufficient sample quantity, only surgical specimens from colon resections were used. Before being sent to each lab, the FFPE samples were reviewed by a pathologist to determine that the sample was of sufficient quality and quantity for testing. We asked each lab to use their standard clinical testing procedures. Clarient uses a polymerase chain reaction (PCR) method for mutation detection, Genzyme uses single nucleotide primer extension assay with fragment analysis by capillary electrophoresis, and the other three labs all use sequencing. Preliminary data (n=11 samples tested to date) suggests good agreement across laboratories despite differences in mutation detection methodologies.

Dataset Information

Competitive amplification of differentially melting amplicons (CADMA) improves KRAS hotspot mutation testing in colorectal cancer.

Publications

Competitive amplification of differentially melting amplicons (CADMA) improves KRAS hotspot mutation testing in colorectal cancer.

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